Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency†

Reduction of Fe(III) to Fe(II) by Fe(III) chelate reductase is thought to be an obligatory step in iron uptake as well as the primary factor in making iron available for absorption by all plants except grasses. Fe(III) chelate reductase has also been suggested to play a more general role in the regulation of cation absorption. In order to experimentally address the importance of Fe(III) chelate reductase activity in the mineral nutrition of plants, three Arabidopsis thaliana mutants (frd1-1, frd1-2 and frd1-3), that do not show induction of Fe(III) chelate reductase activity under iron-deficient growth conditions, have been isolated and characterized. These mutants are still capable of acidifying the rhizosphere under iron-deficiency and accumulate more Zn and Mn in their shoots relative to wild-type plants regardless of iron status. frd1 mutants do not translocate radiolabeled iron to the shoots when roots are presented with a tightly chelated form of Fe(III). These results: (1) confirm that iron must be reduced before it can be transported, (2) show that Fe(III) reduction can be uncoupled from proton release, the other major iron-deficiency response, and (3) demonstrate that Fe(III) chelate reductase activity per se is not necessarily responsible for accumulation of cations previously observed in pea and tomato mutants with constitutively high levels of Fe(III) chelate reductase activity.     

The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.     

Endophytic yeast protect plants against metal toxicity by inhibiting plant metal uptake through an ethylene-dependent mechanism

Toxic metal pollution requires significant adjustments in plant metabolism. Here, we show that the plant microbiota plays an important role in this process. The endophytic Sporobolomyces ruberrimus isolated from a serpentine population of Arabidopsis arenosa protected plants against excess metals. Coculture with its native host and Arabidopsis thaliana inhibited Fe and Ni uptake. It had no effect on host Zn and Cd uptake. Fe uptake inhibition was confirmed in wheat and rape. Our investigations show that, for the metal inhibitory effect, the interference of microorganisms in plant ethylene homeostasis is necessary. Application of an ethylene synthesis inhibitor, as well as loss-of-function mutations in canonical ethylene signalling genes, prevented metal uptake inhibition by the fungus. Coculture with S. ruberrimus significantly changed the expression of Fe homeostasis genes: IRT1, OPT3, OPT6, bHLH38 and bHLH39 in wild-type (WT) A. thaliana. The expression pattern of these genes in WT plants and in the ethylene signalling defective mutants significantly differed and coincided with the plant accumulation phenotype. Most notably, down-regulation of the expression of IRT1 solely in WT was necessary for the inhibition of metal uptake in plants. This study shows that microorganisms optimize plant Fe and Ni uptake by fine-tuning plant metal homeostasis.     

Origins of specificity and cross‐talk in metal ion sensing by Bacillus subtilis Fur

Fur (f erric u ptake r egulator) is the master regulator of iron homeostasis in many bacteria, but how it responds specifically to Fe(II) in vivo is not clear. Biochemical analyses of Bacillus subtilis Fur (BsFur) reveal that in addition to Fe(II), both Zn(II) and Mn(II) allosterically activate BsFur–DNA binding. Dimeric BsFur co‐purifies with site 1 structural Zn(II) (Fur₂Zn₂) and can bind four additional Zn(II) or Mn(II) ions per dimer. Metal ion binding at previously described site 3 occurs with highest affinity, but the Fur₂Zn₂:Me₂ form has only a modest increase in DNA binding affinity (approximately sevenfold). Metallation of site 2 (Fur₂Zn₂:Me₄) leads to a ～ 150‐fold further enhancement in DNA binding affinity. Fe(II) binding studies indicate that BsFur buffers the intracellular Fe(II) concentration at ～ 1 μM. Both Mn(II) and Zn(II) are normally buffered at levels insufficient for metallation of BsFur site 2, thereby accounting for the lack of cross‐talk observed in vivo. However, in a perR mutant, where the BsFur concentration is elevated, BsFur may now use Mn(II) as a co‐repressor and inappropriately repress iron uptake. Since PerR repression of fur is enhanced by Mn(II), and antagonized by Fe(II), PerR may co‐regulate Fe(II) homeostasis by modulating BsFur levels in response to the Mn(II)/Fe(II) ratio.     

Abscisic acid alleviates iron deficiency by promoting root iron reutilization and transport from root to shoot in Arabidopsis

Abscisic acid (ABA) has been demonstrated to be involved in iron (Fe) homeostasis, but the underlying mechanism is largely unknown. Here, we found that Fe deficiency induced ABA accumulation rapidly (within 6 h) in the roots of Arabidopsis. Exogenous ABA at 0.5 μM decreased the amount of root apoplastic Fe bound to pectin and hemicellulose, and increased the shoot Fe content significantly, thus alleviating Fe deficiency‐induced chlorosis. Exogenous ABA promoted the secretion of phenolics to release apoplastic Fe and up‐regulated the expression of AtNRAMP3 to enhance reutilization of Fe stored in the vacuoles, leading to a higher level of soluble Fe and lower ferric–chelate reductase (FCR) activity in roots. Treatment with ABA also led to increased Fe concentrations in the xylem sap, partially because of the up‐regulation of AtFRD3, AtYSL2 and AtNAS1, genes related to long‐distance transport of Fe. Exogenous ABA could not alleviate the chlorosis of abi5 mutant resulting from the significantly low expression of AtYSL2 and low transport of Fe from root to shoot. Taken together, our data support the conclusion that ABA is involved in the reutilization and transport of Fe from root to shoot under Fe deficiency conditions in Arabidopsis.     

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less ⁶²Zn-DMA than ⁶²Zn²⁺. Importantly, supply of ⁶²Zn-DMA rather than ⁶²Zn²⁺ increased the translocation of ⁶²Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

FRD3, a member of the multidrug and toxin efflux family,controls iron deficiency responses in Arabidopsis

We present the cloning and characterization of an Arabidopsis gene, FRD3, involved in iron homeostasis. Plants carrying any of the three alleles of frd3 constitutively express three strategy I iron deficiency responses and misexpress a number of iron deficiency-regulated genes. Mutant plants also accumulate approximately twofold excess iron, fourfold excess manganese, and twofold excess zinc in their shoots. frd3-3 was first identified as man1. The FRD3 gene is expressed at detectable levels in roots but not in shoots and is predicted to encode a membrane protein belonging to the multidrug and toxin efflux family. Other members of this family have been implicated in a variety of processes and are likely to transport small organic molecules. The phenotypes of frd3 mutant plants, which are consistent with a defect in either iron deficiency signaling or iron distribution, indicate that FRD3 is an important component of iron homeostasis in Arabidopsis.     

The arbuscular mycorrhizal fungus Rhizophagus irregularis uses a reductive iron assimilation pathway for high‐affinity iron uptake

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high‐affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1‐2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane‐localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe‐deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe‐limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.